Copper is an essential trace element, which is critical to human health. It serves as a cofactor for many fundamental biological reactions, and is required in processes such as respiration, superoxide disproportionation, degradation of amines, or for iron mobilization and uptake. Because free copper ions catalyze the production of highly reactive hydroxyl radicals, which can damage lipids, proteins, DNA, and other biomolecules, copper uptake, distribution, and incorporation into proteins require a sophisticated machinery. Because the extracellular availability of copper may vary over time, cells are required to maintain buffer sites not only as a defense against deficiency, but also as protection from abnormally high levels. While great progress has been made in understanding the mechanisms of copper uptake, distribution, and regulation on a molecular level, still little is known about the subcellular location of such temporal storage sites and their redistribution during normal cell function or in specific disease states. To study these fundamental processes, new techniques are required for visualizing the dynamics of cellular copper in context of a live cell and to identify associated organelles or compartments. The goal of this grant application is to develop tools that will enable biologists to visualize cellular copper by combining two powerful imaging modalities, light and X-ray fluorescence microscopy. The former approach entails the development of membrane diffusible high-contrast- ratio copper-responsive fluorescent probes that will be suitable for interrogating the subcellular location and dynamics of kinetically labile copper pools in live cells. The second imaging modality is a highly sensitive synchrotron-based microanalytical technique that will provide quantitative information about the distribution of total copper and other transition elements in fixed cells. In order to bridge the two complementary techniques, we will develop a catalytically amplifiable xenobiotic fluorescent labeling approach, thus enabling direct correlative light and X-ray fluorescence microscopy of copper and associated cellular structures. Both imaging techniques will be applied to elucidate the role of the Golgi apparatus in copper redistribution and inheritance during mitosis and cell proliferation. The developed tools are expected to be of critical importance for elucidating other aspects of copper homeostasis and for the long-term development of novel diagnostic and therapeutic tools that will aid in combating copper related human diseases. PUBLIC HEALTH RELEVANCE: Copper is an essential trace element, which is critical to human health. An increasing number of diseases, including Wilson disease, Menkes syndrome, or Alzheimer's disease, are caused by impaired copper transport and regulation. We propose the development of sensitive probes and techniques that will be suitable to visualize copper by light and X-ray fluorescence microscopy, as these tools are expected to significantly aid and advance studies of copper storage and regulation, thus help unravel the fundamental mechanisms of these diseases.